In order to be useful as pharmaceutical preparations, bioactive agents must be able to reach the therapeutic site in an adequate therapeutic effective amount. While many bioactive agents and drugs are stable in vivo, others are often degraded. When such degradation occurs prior to the drug or bioactive agent reaching its target site, a non-therapeutic amount of drug will reach the target site. Other drugs or bioactive agents are taken up by non-target systems, once again resulting in the lack of a therapeutic amount of a drug or bioactive agent reaching the target site at therapeutically effective amounts. Certain polar drugs can not enter cells at all because of their inability to cross the target cell membrane. The only way that these polar drugs may enter a cell is by uptake by the process of endocytosis, exposing them to degradative lysosomal enzymes in the cell. Yet another problem in the therapeutic delivery of drugs or bioactive agents is the inability to administer a high enough concentration of the drug or bioactive agent to be therapeutic, while avoiding toxicities often associated with some drugs or bioactive agents. These problems have been approached by a number of different methods. When a drug or bioactive agent has no toxicity associated with it, it may be administered in high enough doses to account for degradation, removal by non-target organs and lack of targeting to the site where the therapeutic drug or bioactive agent is required. However, many drugs or bioactive agents are either too expensive to allow such waste or have toxicities that prevent administration of such high dosages. Numerous methods have been used to overcome some of the problems encountered in administering therapeutic amounts of drugs or bioactive agents.
One such method is the encapsulation of drugs or bioactive agents in liposomes. While some drugs or bioactive agents can be encapsulated in liposomes at therapeutically effective doses by passive loading or by gradient loading, these methods are limited either to drugs or bioactive agents with specific chemical properties or to drugs or bioactive agents that can be administered in relatively low concentrations. Some bioactive compounds such as weak bases or weak acids can be loaded remotely into preformed liposomes to form highly concentrated complexes. This type of loading, referred to as remote or gradient loading, requires that the drug or bioactive agent be temporarily able to pass through the lipid bilayer of the liposome. However, this is not the case for all bioactive molecules, many of which cannot pass through the liposomal bilayer.
One area in which attempts to administer therapeutic levels of drugs or bioactive agents have been only partially successful is the area of gene therapy. Gene therapy involves the introduction of an exogenous gene into an appropriate cell type, followed by enablement of the gene's expression within the cell at therapeutically relevant levels. Such therapy has progressed, in a relatively short period of time, from basic research to the introduction into cells of a variety of genes, including those useful for treating cancers (Duque et al., Histol Histopathol, 13: 231-242 (1998); Runnebaum et al., Anticancer Res., 17: 2887-2890 (1997)). While naked DNA, in some cases has been taken up into cells (Wolff et al., Science, 247: 1465-1468 (1990)), it generally cannot be, due to its large size and high degree of negative charge; moreover, naked DNA cannot be designed so as to be targeted to specific cells. Accordingly, successful gene therapy generally is reliant upon the availability of “vectors” for introducing DNA and other nucleic acids into cells.
Presently, there are two major groups of DNA delivery systems, viral and non-viral. Viral vectors, including replication-deficient viruses, such as retroviruses, adenoviruses, and adeno-associated viruses, have thus far been the most widely described gene delivery vehicles (Robbins et al., Trends in Biotech, 16: 3540 (1998)). However, their use has been hampered by the immunogenicity of their viral components, potential risk of reversion to a replication-competent state, potential introduction of tumorgenic mutations, lack of targeting mechanisms, limitations in DNA capacity, difficulty in large scale production and other factors (see, e.g., Lee and Huang, J Biol Chem, 271: 8481-8487 (1996)).
Two major types of nonviral vehicles have been developed as alternatives to viral vectors. Cationic liposome-DNA complexes (or “lipoplexes,” Feigner et al., Proc Natl Acad Sci USA, 84: 7413-7417 (1987)), consisting of cationic lipids and DNA have thus far been the most widely described alternative to viral vectors for gene delivery. However, such lipoplexes suffer from several major drawbacks when used in gene therapy, including low stability, high cytotoxicity, non-biodegradability, poor condensation and protection of DNA, serum sensitivity, large size and lack of tissue specificity. Moreover, as the lipoplexes are positively charged, they generally interact nonspecifically with the negatively charged surfaces of most cells; accordingly, it is generally not possible to target such lipoplexes to specific sites in vivo.
Another variation of lipoplexes and DNA involves polylysine-condensed DNA bound to anionic liposomes (Lee and Huang, J Biol Chem, 271: 8481-8487 (1996)). These require certain anionic lipids to form the active structure. The lipoplexes formed either do not completely encapsulate the DNA or must form two or more bilayers around the condensed DNA. In the latter case delivery to the cytoplasm would require the DNA to cross at least three membranes. This would be expected to inhibit transfection efficiency. In the former case, stability may be compromised by exposure of the DNA in physiological salt solutions.
Liposomes are an additional type of nonviral vector alternative, and offer several advantages for such use in comparison to the lipoplexes. For example, liposomal bilayers form around encapsulated nucleic acids, thereby protecting the nucleic acids from degradation by environmental nucleases; lipoplexes, by contrast, do not encapsulate nucleic acids, and hence, cannot completely sequester them away from environmental nucleases. Moreover, liposomes can encapsulate, in their aqueous compartments, other bioactive agents in addition to nucleic acids; lipoplexes, by contrast, cannot because they do not encapsulate aqueous volume. Furthermore, liposomes can be made to be neutrally charged or anionic, as opposed to the restricted ionic nature of the aforementioned lipoplexes. Thus, liposomes can be designed so as to avoid cytotoxicities induced by the delivery vehicle itself and to enhance their accumulation at specific sites of interest.
While the concept of encapsulating bioactive agents in liposomes is not new, many agents have been difficult to encapsulate in liposomes at any level and others have proven difficult to encapsulate in liposomes at levels that would be therapeutically effective. Many small molecules can be encapsulated in liposomes but leak out. Thus, it has also been difficult to encapsulate some bioactive agents and have them retained within the liposomes at a therapeutically effective dose for a therapeutically effective time. For instance, it has been difficult to encapsulate particularly large molecules into a complex within a liposome. It has also been difficult to use many water soluble molecules as therapeutic agents because they are unable to penetrate the cell membrane. When encapsulated stably into liposomes that can fuse to cell membranes, it is possible to deliver these drugs at therapeutically effective doses into the target cells. The method of the present invention enables formation of liposomes containing such drugs or bioactive agents in a therapeutically useful form.
Several attempts have been made to encapsulate nucleic acids in liposomes, these including use of the reverse-phase evaporation (Fraley et al., J Biol Chem, 255: 10431-10435 (1980)), dehydration-rehydration (Alizo et al., J Microencap, 7: 497-503 (1990)) and freeze-thaw (Monnard et al., Biochem Biophys Acta, 1329: 39-50 (1997)) methods of liposome formation. However, each of these methods has several limitations, including requirements for low starting concentrations of nucleic acid, resulting in significant percentages of empty vesicles in the product liposomes, inability to reproducibly encapsulate sufficient quantity of DNA in liposomes to be therapeutically effective at the desired target site and difficulties in optimizing the vehicles for protection of their encapsulated nucleic acids from nuclease-mediated degradation.
Attempts have also been made to complex DNA with complexing agents and subsequently encapsulate the complexed DNA in liposomes. Complexing agents are agents that react with other molecules causing the precipitation or condensation of the molecules. Complexing agents useful in the practice of the present invention are selected from the group consisting of charged molecules that have a charge opposite to the charge on the bioactive agent. The complexing agent may be selected from the group of charged molecules consisting of spermine, spermidine, hexammine cobalt, calcium ions, magnesium ions, polylysines, polyhistidines, protamines, polyanions such as heparin and dextran sulfate, citrate ions, or sulfate ions. For instance, polycations of charge +3 or higher, e.g., polyamines, polylysine and hexammine cobalt (III) are known (see Chattoraj et al., J Mol Biol, 121: 327-337 (1978); Gosule L C and Schellman J A. Nature 259: 333-335 (1976); Vitello et al., Gene Therapy, 3: 396-404 (1996); Widom et al. J. Mol. Biol., 144: 431-453 (1980); Arscott et al., Biopolymers, 30: 619-630 (1990); Wilson et al., Biochem, 18: 2192-2196 (1979)) to be able to condense DNA molecules, through interaction with multiple negative charges on the DNA. Polyamines, e.g., spermidine (3+) and spermine(4+), have, unlike other types of polycations, been found to occur naturally in all living cells (see, e.g., Ames and Dubin, J Biol Chem, 253: 769-775 (1960); Tabor and Tabor, Annu Rev Biochem, 53: 749-790 (1984)). High polyamine levels are known to exist in actively proliferating animal cells, and are believed to be essential therein for maintaining normal cell growth (Ames and Dubin, J Biol Chem, 253: 769-775 (1960); Tabor and Tabor, Annu Rev Biochem, 53: 749-790 (1984); Hafner et al., J Biol Chem, 254: 12419-12426 (1979); Pegg, Biochem J, 234: 249-262 (1986)).
Liposome encapsulation of spermine-condensed linear DNA in liposomes has been attempted by Tikchonenko et al., Gene, 63: 321-330 (1988). However, the starting DNA concentration therein was low, with the consequence that the resulting liposomes also had a low ratio of encapsulated DNA to liposomal lipid (0.02-0.2 micrograms DNA per micromole lipid). Moreover, such condensation of linear DNA molecules in the absence of intermolecular DNA aggregation required control over spermine concentrations to an impracticable degree of precision. Additionally, Baeza et al., Ori Life Evol Biosphere, 21: 225-252 (1992) and Ibanez et al., Biochem Cell Biol, 74: 633-643 (1996) both report encapsulation of 1-4 micrograms per micromole of spermine-condensed SV40 plasmid DNA in liposomes. However, neither of their preparations were dialyzed against high salt buffers subsequent to liposome formation, the reported amounts of encapsulated DNA actually may include a significant percentage of unencapsulated DNA. Since these liposomal formulations were not exposed to DNAase degradation to determine the percentage of DNA actually sequestered in the liposomes, the high reported amounts probably do not reflect actually encapsulated DNA.
Efficient preparation and use of liposomal encapsulated nucleic acids requires the use of high-concentration suspensions of nucleic acids, in order to minimize the percentage of empty liposomes resulting from the process and to maximize the DNA:liposomal lipid ratios. However, condensation of DNA at high concentrations during known methods of liposome formation generally results in intermolecular aggregation, leading to the formation of nucleic acid-based structures unsuitable for gene delivery. Large aggregates formed by condensation of DNA directly with a complexing agent cannot be easily encapsulated in liposome and such large aggregate structures (on the order of the size of cells) can not efficiently deliver materials to target cells. For instance, if the aggregates are larger than 500 nm, they are rapidly cleared from the circulation because of their size after intravenous administration. On the other hand, larger aggregates may be administered to cells in vitro. However, sometimes the aggregates as too large too be taken up by cells.
Thus, in order to deliver a variety of drugs in therapeutically effective amounts into target cells, it was necessary to provide a method of making liposomes that contain bioactive agents complexed so as to decrease their permeability through the lipid bilayer, while providing a method that also limits the size of the complex to be encapsulated in the liposome so that the resultant therapeutic product is in a therapeutic size range.